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ANTI-ANAEMIC POTENTIAL… Kasang et al FJS

FUDMA Journal of Sciences (FJS) Vol. 4 No. 3, September, 2020, pp 370 - 374 370

ANTI-ANAEMIC POTENTIAL OF METHANOLIC LEAF EXTRACT OF MUCUNA PRURIENS ON

PHENYLHYDRAZINE (PHZ) INDUCED ANAEMIC ALBINO WISTAR RATS.

*1Kasang Naman, 1Habibat Oseni and 2Emmanuel Enoh

1Department of Biological Science, Kaduna State University, Tafawa Balewa Way, Kaduna, Kaduna State, Nigeria.

2Nigerian Institute for Tryponosomiasis Research (NITR) Kaduna State, Nigeria

Corresponding Author’s email: [email protected] 08065667859

ABSTRACT

The antianaemic potential of methanolic leaf extracts of Mucuna pruriens was investigated using

phenylhydrazine (PHZ) induced anaemic albino Wistar rats. Fifteen rats used for the study were randomized

into five experimental groups. To induced anaemia, the rats (except the normal control, Group E), received 60

mg/kg of the haemolytic agent Phenylhydrazine intraperitoneally (i.p) for two consecutive days. Anaemic

Wistar rats in groups A and B received a daily oral dose of 500 and 250 mg/kg of the methanolic leaf extract

of Mucuna pruriens. Nweze et al. (2016) had reported a median lethal dose greater than 5000 mg/kg for the

methanol leaf extract of Mucuna pruriens. Groups C and D received Vitamin B12 (10 mg/kg) and normal

saline (1 ml/kg), respectively. Normal control rats also received normal saline (1 ml/kg). Extract or normal

saline was administered per os (p.o) while vitamin B12 was administered i.p. for a duration of 21 days.

Packed cell volume (PCV) and haemoglobin concentration were determined weekly for three weeks. The

result of the study indicated that both the methanolic leaf extract of Mucuna pruriens and Vitamin B12

significantly (p < 0.05) increased the packed cell volume and haemoglobin concentrations in treated rats

compared to the negative control group of rats. This indicated that the methanolic leaf extract of Mucuna

pruriens has anti-anaemic properties and could be utilized in the management of anaemia.

Keywords: Phenylhydrazine, Anaemia, Anti-anaemic properties, Vitamin B12, Mucuna pruriens.

INTRODUCTION Mucuna pruriens is a wild tropical legume native to Africa

and Asia (Duke 1981 in Nweze et al., 2017), it belong to the

family Fabaceae, and is among the various under-utilized wild

legumes. Mucuna pruriens is commonly called ‘velvet bean’,

‘cowhage’ or ‘cowitch’ in English, Eni in Hausa, Agbala or

Agbaloko in Ibo and Werepe in Yoruba. The plant is known for

the intense irritation and itching, it produces on contact with

the skin when the fruit is mature and dries (Andersen et al.,

2015). The itch is caused by mucunain and serotonin contained

in the hairs lining the seed pods called trichomes (Reddy et al.,

2008).

It has nutritional potential as a rich source of protein (23-35%)

(Teixeira et al., 2003), which is comparable to those of other

pulses such as soybean, rice bean and lima bean (Gurumoorthi

et al., 2003). In South East Asia, the immature pods and leaves

of mucuna beans are used as vegetables. The ethnic groups of

Eastern Nigeria use the seeds of Mucuna sloanei and Mucuna

puriens as a condiment or as garnishing for the main dish

(Ukachukwu et al., 2000). All parts of M. pruriens has been

reported to possess valuable medicinal properties and it has

been investigated in various contexts. The seeds have laxative,

anthelmintic, alexipharmic and tonic effects (Taylor, 2005).

The L-dopa content of the seeds is responsible for its efficacy

in the management of Parkinson’s disease (Manyam et al.,

2004). The dried crushed root is applied for relief of toothaches

(Hishika et al., 1981). The leaves possess aphrodisiac and

antimicrobial activities (Warrier et al., 1996) and are used in

the treatment of ulcers, cephalgia and general debility. It is

reported that people suffering from debilitating disease

conditions, acute blood loss and blood deficiency diseases

consume leaves of M. pruriens (Akindele and Busayo, 2011).

Anaemia is a condition in which haemoglobin (Hb)

concentration and/or red blood cell (RBC) numbers are lower

than normal and insufficient to meet an individual’s

physiological needs (WHO, 2011). Alternately, World health

organization (WHO) defined anaemia as a haemoglobin (Hb)

concentration less than 130g/l in men and 120g/l in women.

Anaemia develops when the rate of red blood cell production

by the bone marrow fails to keep pace with loss or destruction

(Reid et al., 2008). Haemoglobin is the oxygen carrying part of

red blood cells. The role of Hb in carrying oxygen to the

tissues explains the most common clinical symptoms of

anaemia, which include fatigue, shortness of breath, bounding

pulses or palpitations, and conjunctival and palmar pallor

(WHO, 2011)

Approximately one-third of the world’s population (32.9%)

was estimated to suffer from anaemia in 2010 (Kassebaum et

al., 2014). The population groups most vulnerable to anaemia

as of 2016 include: (42%) children under 5 years of age,

particularly infants and children under 2 years of age; (39%);

women of reproductive age; and (46%) pregnant women

(WHO, 2016).

Anaemia is one of the most widespread public health problems,

especially in developing countries due to nutritional problems

(such as iron or vitamin deficiency), low socioeconomic status,

inherited disorders, recurrent infections and an increased

prevalence of blood parasites especially Plasmodium,

Trypanosomes and helminthic infestation (Sanni et al., 2005).

FUDMA Journal of Sciences (FJS)

ISSN online: 2616-1370

ISSN print: 2645 - 2944

Vol. 4 No. 3, September, 2020, pp 370 – 374

DOI: https://doi.org/10.33003/fjs-2020-0403-399

mailto:[email protected]

https://doi.org/10.33003/fjs-2020-0403-399



It has an important health, welfare, social, and economic

consequences, these include impaired cognitive development,

reduced physical work capacity and in severe cases increased

risk of mortality, particularly during the perinatal period (Van

den Broek et al., 2000).

In the tropics, due to prevalence of malaria and other parasitic

infections, between10 to 20% of the population is reported to

have less than 100 g/l of haemoglobin in the blood (Diallo et

al., 2008). The great loss in terms of clinical diagnosis and

treatment and even depletion in human resources as a result of

anemia could be prevented with adequate exploration into

medicinal plant that can naturally boost blood level. Hence,

this study investigated the haematological effects of Methanol

leaf extract of Mucuna pruriens on Phenylhydrazine (PHZ)

induced anaemic Wistar strain of albino rats.

MATERIALS AND METHODS

Plant Collection and Identification

The fresh leaves of velvet bean plant were collected locally

from Zaria and identified by Mallam Galla at the Herbarium of

Kaduna State University.

Preparation of the Methanolic Plant Extract The leaves of M. pruriens were washed and air dried at room

temperature. Then it was pulverized and macerated in 2 L of

methanol for 72 hrs.The mixture was decanted through a glass

funnel fitted with Whatmann No. 1 filter paper. The filtrate was

then allowed to evaporate to dryness via open air evaporation.

The dried extract was afterwards weighed and stored until

when required.

Experimental Animals

Wistar rats of both sexes and weighing between 70g and 130 g

were purchased from the animal house unit of the Nigerian

Institute for Trypanosomiasis Research (NITR) Kaduna State.

The animals were housed in locally fabricated cages within the

Zoology Laboratory of Department of Biological Sciences

Kaduna State University and were allowed to a seven days

acclimatization period before the commencement of the study.

The experimental animals were maintained on animal feed and

given water ad libitum. Beddings were changed twice weekly.

Haematological Parameters

Before administration of phenylhydrazine in the rats, the

baseline values of two heamatological parameters were

recorded, namely packed cell volume and haemoglobin

concentration. Packed Cell Volume (PCV) was determined by

the Microhaematocrit method, while the Haemoglobin (Hb)

concentration of each animal was estimated by

Cyanomethaemoglobin method using a spectrophotometer.

Anti-anaemic activity

For the evaluation of anti-anaemic effect of the plant extract,

the methods of Joshi et al. (2018) and Gupta et al. (2018) were

adopted with some modifications. Briefly, anemia was induced

by intra-peritoneal injection of phenylhydrazine at 60 mg/kg

body weight (Pandey et al., 2016; Ragupathy, 2008). Rats that

developed anaemia with PCV level below 39% and

Haemoglobin concentration lower than 13 g/dl were recruited

for the study. The anaemic rats were divided into five groups of

three (3) rats each as follows:

Group 1: (Phenylhydrazine + Low dose extract, 250 mg/kg);

Group 2: (Phenylhydrazine + High dose extract, 500 mg/kg);

Group 3: (Phenylhydrazine +10 mg/kg Vitamin B12 – positive

control);

Group 4: (Phenylhydrazine + 1ml/kg normal saline - negative

control);

Group 5: (non-anemic + 1ml/kg normal saline - Normal

control).

The dose of the extract was based on the work of Nweze et al.

(2016) who reported a median lethal dose (LD50) greater than

5000 mg/kg for the methanol leaf extract of Mucuna pruriens.

Treatment with methanol leaf extract of M. pruriens, vitamin

B12, or normal saline was given orally throughout the duration

of the study.

STATISTICAL ANALYSIS

The results of the haematological estimations were presented as

mean ± SD of three animals per group. Total variations present

in a set of data were estimated by One Way Analysis of

Variance (ANOVA) and p value less than 0.05 was considered

statistically significant.

RESULTS

Prior to the administration of phenylhydrazine (PHZ), the

Packed cell volumes (PCV) of rats were between 49 and 55 %

(Table 1), with no significant difference (p > 0.05) in the mean

PCV values of rats in the various experimental groups. Upon

administration of phenylhydrazine (60 mg/kg body weight)

there was an acute decline in mean PCV with values ranging

from 31-35 percent in all the experimental groups except in the

normal control. Between the PHZ treated groups, there was no

statistically significant difference in the packed cell volumes in

all the anaemic rats (p > 0.05). However, the mean packed cell

volumes of anaemic rats were significantly lower (p < 0.05)

than that of the normal control group (Table 1).

On day 21, the pattern of changes in the haematological

parameters under investigation amongst group 3 (PHZ +

10mg/kg vitamin B12: positive control) rats were observed to

be essentially similar to the pattern obtained amongst group 1

(PHZ + low dose extract) rats and group 2 (PHZ + high dose

extract). Furthermore, when the extract treated groups were

compared with the Normal control (Group 5), there was no

significant difference (p > 0.05) with PCV values of the extract

treated group approaching those of normal control. However,

when compared with group 4 (PHZ + 1ml/kg normal saline:

negative control) there was significant difference (p < 0.05)

with elevations in PCV among the extract treated groups.



Table 1: Effect of plant extracts on packed cell volume of phenyl hydrazine induced anaemic rats

Treatment groups Baseline D0 D7 D14 D21

PHZ + 250 mg/kg extract 49.6±3.0a 32.0±3.46a 40.7±4.51b 42.9±2.11c 53.6±2.15b

PHZ + 500 mg/kg extract 51.7±2.9a 31.7±2.61a 31.3±1.53a 36.8±0.46b 50.8±0.39b

PHZ + 10 mg/kg Vitamin

B12

49.3±2.0a 33.7±3.22a 44.0±4.51b 48.3±0.19d 56.4±0.03b

PHZ + 1ml/kg normal

saline

52.7±2.5a 35.3±2.08a 27.3±2.0a 23.5±0.32a 38.8±0.46a

Normal control 55.0±3.0a 55.0±1.0b 54.3±2.52c 55.8±3.01e 55.2±1.41b

p value 0.2210 0.0023* 0.0345* 0.0026* 0.0138*

Values are given as mean ± standard error of mean (SEM). In each column, values with differing superscripts have statistically

significant difference (p < 0.05)

The effect of treatment with methanolic leaf extract of Mucuna

pruriens on Haemoglobin Concentration (Hb) of rats is shown

in Table 2. Haemoglobin Concentration of anaemic rats treated

with 250 and 500 mg/kg of the methanolic leaf extract recorded

steady increase in mean Hb concentration compared to group 4

(PHZ + 1ml/kg normal saline: negative control). At Day 21, the

Hb values of rats that received the extracts and Vitamins B12

had return to normal values, resulting to no significant

difference in the mean values when compared with the normal

control group (p > 0.05). But when compared with group 4

(PHZ + 1ml/kg normal saline: negative control) there was high

significant difference (p < 0.05).

Table 2. Effect of plant extracts on haemoglobin concentration in phenyl hydrazine induced anaemic rats

Treatment groups Baseline D0 D7 D14 D21

Anaemic + 250 mg/kg

extract

16.5±1.8a 10.7±1.1a 13.6±0.6b 14.3±1.6bc 17.9±2.3b

Anaemic + 500 mg/kg

extract

17.2±1.6a 10.6±0.9a 10.4±0.3a 12.3±0.2b 16.9±0.4b

Anaemic + 10 mg/kg

Vitamin B12

16.4±0.2a 11.2±0.1a 14.7±0.7b 16.1±2.1cd 18.8±0.6b

Anaemic + 1ml/kg

normal saline

17.6±0.4a 11.8±0.5a 9.1±1.3a 7.83±0.6a 12.8±1.2a

Normal control 18.3±1.15a 18.3±0.2b 17.8±1.1c 17.6±1.5d 18.4±1.7b

p value 0.1262 0.0328* 0.0023* 0.0007* 0.0007*

Values are given as mean ± standard error of mean (SEM). In each column, values with differing superscripts have statistically



significant difference (p < 0.05)

DISCUSSION

The results of this study indicate that phenylhydrazine induced

anaemia in rats. These findings agree with those of Dholi et al.

(2016), Droucoula et al. (2017) and Umaru et al. (2018) who

also reported that administration of PHZ induces acute anaemia

in rats. Phenylhydrazine induces haemolytic anaemia though

oxidative alternations to red cell membrane proteins resulting

in red cell injury and lysis (Berger, 2007).

The elevations in haematological parameters recorded in

anaemic rats treated with either Vitamin B12 or the leaf extract

of Mucuna pruriens are indicative of their haematopoietic

properties. Vitamin B12 (cobalamin) is an essential nutrient

required in blood cell formation and maturation. When in

insufficient quantity, vitamin B12 deficiency results in

Megaloblastic anaemia, a condition characterized by

proliferation of immature red cell with reduced lifespan

(Castellanos-Sinco et al., 2015; Koury and Ponka, 2004). There

anti-anaemic activity demonstrated with the doses of the

methanol leaf extract of Mucuna pruriens agrees with those of

Obioma et al. (2014) who investigated the anti-anaemic

potentials of the aqueous extract and raw leaves of Mucuna

pruriens. Similarly, Madukwe et al. (2014) also reported that

the fresh and shade-dried leaf extracts of Mucuna pruriens

significantly increased haemoglobin, packed cell volume and

white cell counts in anaemic rats. In a review article, Ravindra

and Ashvini (2019) also mentioned Mucuna pruriens (Agbala)

as a plant having anti-anaemic potentials. Furthermore, Nweze

et al. (2017) reported that the fresh leaves of M. pruriens are

washed and macerated in water to make a decoction which is

drunk to boost blood supply. The anti-anaemic activity as

demonstrated by Mucuna pruriens may be due to the presence

of blood forming moieties in the leaf extract. Proximate

analysis of the leaves showed the presence of iron, vitamin C

and pro-vitamin A (Akomas et al., 2014; Madukwe et al.,

2014). Iron is an essential haematopoietic factor required in the

formation of the haem component of haemoglobin found in red

cells. Vitamin C has also been reported to participate in

haematopoiesis (Musyoka et al., 2016).

Phytochemical screening of the leaf extracts has also revealed

the presence of saponins, flavonoids and alkaloids (Nweze et

al., 2016). Certain class flavonoids and alkaloids have been

reported to possessing antioxidant activity. Thus, the presence

of these antioxidants in the leaf extract of M. pruriens may

facilitate the reversal of the damaging effect of phenyl

hydrazine on red cells. Phenyl hydrazine is reported to induce

anaemia through oxidative damage to red cell membrane by

increasing the formation of reactive oxygen species (Lavanya

et al., 2018). However, alkaloids and flavonoids protect cells as

powerful antioxidants which prevent or repair damage done to

red cells by free radicals or highly reactive oxygen species

(Beack et al., 2020).

In conclusion, the results of the analysis carried out on

Phenylhydrazine (PHZ) induced anaemia in Wistar strain of

albino rats orally treated with methanolic leaf extract of M.

pruriens revealed that the extracts boosted blood production,

thereby suggesting its antianaemic potentials which can be

used in folklore medicine for the management of anaemia.

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